Abstract
Previous research has implicated negative regulators of cell cycle in Escherichia coli, in particular the SeqA protein, in the stringent response. The stringent response is caused by amino acid starvation, and results in a complete cessation of initiation of replication at the origin of replication, oriC, causing a percentage of a population of cells to arrest with 1N chromosome equivalents. We believe that long-term stable binding of SeqA to hemi-methylatcd GATC sites at oriC during stringent response is the cause of this arrest. Mutations that affect the capacity of Seq A to bind at the origin also remove the ability of the cells to arrest at 1N. Deletion of seqA from the E. coli genome causes deficiency in stringent arrest. Deletion of dam methylase — which leads to unmethylated DNA, to which SeqA does not bind — causes the same effect. Over-expression of dam, which causes premature full methylation at newly replicated GATC sites, also disrupts stringent arrest. Mutation of 9 GATC sites in oriC to GTTC also partially disturbs stringent arrest. We are also interested in the role of RNA polymerase in facilitating the stringent response. It is known that widespread changes in transcriptional patterns occur during stringent arrest, and we are in possession of strains mutant in the RNA polymerase beta subunit that mimic stringent transcriptional patterns in the absence of amino acid starvation. The rpoB mutants display partial IN and 2N arrest without amino acid starvation. In addition, the mutations described above (seqA, dam, oriCm3) that disrupt stringent arrest also suppress cell cycle arrest phenotypes in rpoB mutants.